U.S. patent number 7,186,697 [Application Number 08/822,033] was granted by the patent office on 2007-03-06 for nucleic acid delivery system, methods of synthesis and use thereof.
This patent grant is currently assigned to Dana-Farber Cancer Institute. Invention is credited to Si-Yi Chen, Wayne A. Marasco.
United States Patent |
7,186,697 |
Marasco , et al. |
March 6, 2007 |
Nucleic acid delivery system, methods of synthesis and use
thereof
Abstract
A nucleic acid delivery system is described. The delivery system
contains a fusion protein having a target moiety and a nucleic acid
binding moiety, and a nucleic acid sequence bound to the nucleic
acid binding moiety of the fusion protein. The target moiety can be
an antibody or a ligand. The use of this nucleic acid delivery
system to transienntly or stably express a desired nucleic acid
sequence in a cell is disclosed. Also disclosed is the use of this
delivery system to target a cell and deliver a desired product.
Inventors: |
Marasco; Wayne A. (Wellesley,
MA), Chen; Si-Yi (Brookline, MA) |
Assignee: |
Dana-Farber Cancer Institute
(Boston, MA)
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Family
ID: |
22736089 |
Appl.
No.: |
08/822,033 |
Filed: |
March 24, 1997 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040023902 A1 |
Feb 5, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08199070 |
Feb 22, 1994 |
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Current U.S.
Class: |
514/44R; 530/350;
536/23.53; 536/23.4; 530/402; 424/178.1 |
Current CPC
Class: |
C12N
15/87 (20130101); C07K 14/46 (20130101); C12N
15/62 (20130101); A61P 35/00 (20180101); C07K
19/00 (20130101); A61K 48/00 (20130101); A61K
47/6807 (20170801); C07K 2319/00 (20130101); C07K
2319/02 (20130101); C07K 2319/80 (20130101); C07K
2319/55 (20130101) |
Current International
Class: |
A61K
31/70 (20060101); A61K 39/395 (20060101); C07H
21/02 (20060101); C07H 21/04 (20060101); C07K
1/00 (20060101) |
Field of
Search: |
;514/2,44
;530/387.1,350,358,387.3 ;536/23.1,23.4,23.5,23.7,24.1
;435/172.3,69.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2012311 |
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Sep 1990 |
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CA |
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WO92/22332 |
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Dec 1992 |
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WO |
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WO93/04701 |
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Mar 1993 |
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WO |
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WO94/02610 |
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Feb 1994 |
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WO |
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WO94/04696 |
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Mar 1994 |
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WO |
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Other References
DT Curiel et al (1991) Proc Natl Acad Sci USA. 88:8850-8854. cited
by examiner .
VK Chaud havy et al (1990) Proc Natl Acad Sci USA 87: 1066-1070.
cited by examiner .
K Ryder et al (1989) Proc Natl Acad Sci USA 86: 1500-1503. cited by
examiner .
Chen, S.Y., et al., Gene Therapy, vol. 2 No. 116-123 (Mar. 2,
1995). cited by other .
Kabanov, A., et al., Journal of Controlled Release 28:15-35 (Jan.
1994). cited by other.
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Primary Examiner: Woitach; Joseph
Attorney, Agent or Firm: Peabody LLP; Nixon Eisenstein;
Ronald I. Karttunen; Leena H.
Parent Case Text
This application is a continuation of application Ser. No.
08/199,070 filed on Feb. 22, 1994 now abandoned.
Claims
We claim:
1. A nucleic acid delivery system comprising: (1) a fusion protein,
wherein said fusion protein is prepared by recombinant techniques
and contains: (a) an antibody targeting moiety, which will
specifically bind to a site on a target cell, and (b) a binding
moiety which will bind to a nucleic acid segment, and (2) a nucleic
acid sequence comprising the nucleic acid segment and a nucleic
acid sequence of interest, wherein the fusion protein is encoded by
a nucleic acid having no stop codon between the antibody targeting
moiety encoding nucleic acid segment and the nucleic acid segment
encoding the binding moiety which will bind to a nucleic acid
segment.
2. The nucleic acid delivery system of claim 1, wherein the binding
moiety is a protein or the nucleic acid binding domain of a
protein, and the binding moiety is fused to the carboxy portion of
the targeting moiety.
3. The nucleic acid delivery system of claim 2, wherein the binding
moiety is the protein protamine.
4. A nucleic acid delivery system comprising a fusion protein
wherein one portion of the fusion protein comprises an antibody,
which will selectively bind to a desired site on a cell, and the
other portion of the fusion protein comprises a protarnine protein
capable of binding to a nucleic acid segment; and the nucleic acid
segment.
5. The nucleic acid delivery system of claim 4, wherein the nucleic
acid segment is a DNA sequence corresponding to a cytotoxin gene or
a fragment thereof which will encode a cytotoxic protein.
6. The nucleic acid delivery system of claim 5, wherein the nucleic
acid segment encodes at least Domain III of Pseudomonas exotoxin
A.
7. A method of use of a nucleic acid delivery system which
comprises administering an effective amount of the nucleic acid
delivery system of claim 4 to serum containing a target cell, and
contacting the target cell with the nucleic acid delivery system,
whereby the target cell is transfected with the nucleic acid
sequence.
8. A nucleic acid delivery system comprising: (1) a fusion protein,
wherein said fusion protein is prepared by recombinant techniques
and contains: (a) an antibody targeting moiety, which will
specifically bind to a site on a target cell, wherein the antibody
is an antibody to a viral envelope protein, a cellular receptor, or
an extracellular domain of an activated receptor, and (b) a binding
moiety which will bind to a nucleic acid segment, and (2) a nucleic
acid sequence comprising the nucleic acid segment and a nucleic
acid sequence of interest.
9. The nucleic acid delivery system of claim 8, wherein the
antibody is to a viral envelope protein.
10. A nucleic acid delivery system comprising: (1) a fusion
protein, wherein said fusion protein is prepared by recombinant
techniques and contains: (a) an antibody targeting moiety, which
will specifically bind to a site on a target cell, wherein the
antibody is a single chain antibody, a Fab portion of an antibody,
or a (Fab').sub.2 segment and (b) a binding moiety which will bind
to a nucleic acid segment, and (2) a nucleic acid sequence
comprising the nucleic acid segment and a nucleic acid sequence of
interest.
11. The nucleic acid delivery system of claim 8 or 10, wherein the
nucleic acid sequence of interest encodes an antibody, a dominant
negative mutant, an antisense RNA, ribozymes, or a cytotoxic
agent.
12. The nucleic acid delivery system of claim 8 or 10, wherein the
nucleic acid segment comprises a promoter operably linked to a
desired gene in the nucleic acid sequence of interest, wherein said
promoter and gene are flanked by 5' and 3' long terminal repeat
(LTR) regions or inverted terminal repeat (ITR) regions.
13. A method of transforming a target cell which comprises adding
an effective amount of the nucleic acid delivery system of claim 8
or 10 to a medium containing the target cell, and contacting the
target cell with the nucleic acid delivery system, whereby the
target cell is transfected with the nucleic acid sequence.
14. The method of claim 13, wherein the nucleic acid sequence is
RNA.
15. A method of preparing a nucleic acid delivery system which
comprises transforming a cell with a vector containing a DNA
segment which encodes the fusion protein of claim 8 or 10 operably
linked to a promoter, incubating the cell, and collecting the
expressed fusion protein.
16. A method of use of a nucleic acid delivery system which
comprises administering an effective amount of the nucleic acid
delivery system of claim 8 or 10 to serum containing a target cell,
and contacting the target cell with the nucleic acid delivery
system, whereby the target cell is transfected with the nucleic
acid sequence.
17. The method of claim 16, wherein the nucleic acid sequence is
RNA.
18. A method of transforming a target cell in vivo with RNA which
comprises administering the nucleic acid delivery system of claim 8
or 10 to a subject containing the target cell, wherein the nucleic
acid sequence is RNA.
Description
In recent years, a new form of therapy, gene therapy, has been
proposed to treat a variety of maladies including cystic fibrosis
(CF) [Rosenfeld, M. A., et al., Cell 68:143 155 (1992); Rosenfeld,
M. A., et al., Science 252:431 434 (1991), Ferkol, T., et al., J.
Clin. Invest. 92:2394 2400 (1993)], tumors such as retinoblastoma,
diseases caused by infection by a virus such as the human
immunodeficiency virus (HIV), for example HIV-1 infection
[Baltimore, D., Nature 335, 395 396 (1988)], etc. In this form of
therapy, a gene is introduced into cells so that the cells will
express that gene. The gene can positively potentiate the cells,
e.g., supply a missing protein, stimulate the immune system, or it
may act in a negative manner, for instance expressing a viral
inhibitor, which can result in the inhibition of the virus such as
HIV-1 replication and thus infection. Several approaches including
anti-sense RNA, ribozymes and dominant-negative mutants have been
shown to be able to inhibit HIV-1 infection at the cellular level
[Hasseloff, J, et al., Nature 334:585 591 (1988); Von der Krol, A.
R., et al., BioTechniques 6:958 976 (1988); Malim, M. H., et al.,
Cell 58:205 214 (1989); Trono, D., et al., Cell 59:113 120 (1989);
Sullenger, B., et al., Cell 63:601 608 (1990); Green, G., et al.,
Cell 58:215 223 (1989); Buonocore, L., et al., Nature 345:625 628
(1990)]. The intracellular delivery and expression of a human
antibody, such as an anti-gp120 single chain antibody, is able to
inhibit viral replication, etc. For example, the anti-gp120
antibody inhibits HIV-1 envelope glycoprotein maturation and
function [Marasco, W. A., et al., PCT Application No.
PCT/US93/06735, filed July 1993].
However, despite these advances, a major impediment to the
development of gene therapy protocols for treatment and prevention
of malignancies, diseases, etc. using any of these strategies is
the relatively inefficient means to effectively transduce the
desired genes into the desired target cells. Although murine
retroviral vectors have been widely used to transfer gene into
cells, they indiscriminately infect many cell types and limitedly
infect desired targeted cells. In addition, retroviral vectors
contain potentially hazardous viral DNA along with therapeutic
genes. Therefore, these vectors may not be optimal as an efficient
transfer system for the human gene therapy of, for instance, AIDS
[Miller, A. D., Nature 357:455 46 (1992); Eglitis, M. A., et al.
Science 230:1395 1398 (1985); Dizerzak, E. A., et al., Nature
331:35 41 (1988)]. To resolve the problem of specific delivery for
HIV infected cells, defective HIV vectors which can specifically
transfer a gene into HIV susceptible cells have been developed.
[Poznansky, M., et al., J. Virol. 65:532 536 (1991); Shimada, T.,
et al., J. Clin. Invest. 88:1043 1047 (1991)]. However, this
approach may not be practical with all viruses and malignancies.
Further, the theoretical potential of recombinant rescue of the
defective vector, however low, may impede its use.
The delivery and expression of a recombinant gene into cells has
also been achieved using liposomes, lipofectin, and calcium
phosphate-precipitated methods either in vitro or in vivo [Nicolau,
C., et al., Proc. Natl. Acad. Sci. USA 80:1068 1072 (1983);
Brigham, K. L, et al., Am. J. Med. Sci. 298:278 281 (1989); Nabel,
E. G., et al. Science 249:1285 1288 (1990); Benvenisty, N. et al.,
Proc. Natl. Acad. Sci. USA 83:9551 9555 (1986); Chen, S. -Y., et
al., J. Virol. 65:5902 5909 (1991)]. These methods have several
advantages over retroviral systems for gene therapy. Plasmid DNA
constructs containing suitable promoter elements are technically
easier and less time consuming to prepare and test than retroviral
vectors. Plasmid DNAs are more suitable for large-scale preparation
than are the infections retroviruses. Plasmid DNAs can also permit
the delivery of larger-sized segments of DNA than is possible with
retrovirus-based systems. One additional advantage is that plasmid
DNA may exclude deleterious side effects of retroviral vectors such
as virus infection or cancer in a small percentage of patients.
However, the potential for delivery of genes in vivo using these
methods is limited by a lack of cell specificity and
efficiency.
In an attempt to overcome the problem of cell-specific gene
transfer, Wu and Wu, J. Biol. Chem. 262:4429 4432 (1987) have
proposed a chemically coupled receptor-mediated gene transfer
system which uses receptor-mediated endocytosis to carry DNA or RNA
molecule into target hepatocytes or primary hematopoietic cells.
The strategy of this system is based on the fact that such cells
possess unique astalglycoprotein receptors on their surface that
bind and internalize asialoglycoproteins, its ligand. The proteins
(ligands) are preferably coupled to poly-L-lysine which can bind
DNA or RNA to form soluble complexes by a strong, electrostatic
interaction. This system has been reported to transfer genes into
the targeted hepatocytes or primary hematopoietic cells at the
cellular level as well as in animal studies [Wu, G. Y., et al., J.
Bio. Chem. 263:14621 14624 (1988); Zenke, M., et al., Proc. Natl.
Acad. Sci. 87:3655 3659 (1990); Wu, C. Y., et al., J. Biol. Chem.
266:14338 14342 (1991); Curiel, D. T., et al., Proc. Natl. Acad.
Sci. USA 88:8850 8854 (1991); Wagner, E. et al., Proc. Natl. Acad.
Sci. USA 87:3410 3414 (1990); Curiel, D. T., et al., Human Gene
Therapy 2:230 238 (1992)]. However, the overall efficiency of this
method has been reported to be relatively low because endocytosis
is relatively inefficient in that the DNA frequently does not get
out of the endosomial compartment and is ultimately degraded in
lysosomes. Thus, multiple administrations are necessary and
antigenicity of this system can be a problem. Furthermore, the
synthesis of the delivery system is relatively time consuming as
one has to first couple the poly-L-lysine to the asialoglycoprotein
and then subsequently couple the ligand-polylysine complex to the
exogenous DNA. Furthermore, in its typical application, the
exogenous DNA introduced into the cell is not presented in a manner
which is stably incorporated into the chromosome. Thus, expression
is transient. Accordingly, repeated administration is necessary for
this reason also. However, as mentioned the polylysine moiety as an
artificial moiety may trigger an antigenic reaction limiting the
ability to repeatedly use this system.
Another form of therapy that has been proposed is delivering an
already expressed protein to the target cell. In one common form of
cancer therapy, one introduces cytopathic or cytotoxic agents to
the malignant cells in order to kill them. However, care must, be
taken to minimize the harm to healthy tissues and cells. Thus,
strategies have been developed to try to specifically target the
unhealthy cells. The use of immunotoxins is one method of such
therapy. An inmunotoxin is a class of cytotoxic agents consisting
of a toxin protein linked to a monoclonal antibody or a ligand,
which binds specifically to a target on the cell surface [Vitetta,
E. S., et al. Science 238:1098 (1987); Pastan, I., et al., Cell
47:641 (1986); Pastan, I., et al., Science 254:1173 (1992)]. Due to
the predicted specificity for the cell and the potential for
efficacy, this therapy has been predicted to play an important role
in therapy against cancer and various diseases. However, in
practice this has not proven to be the case, Rather, the toxins are
highly antigenic proteins. Neutralizing antibodies against these
toxins typically arise within two weeks after the first exposure,
severely limiting their effectiveness after only one or two therapy
sessions. Thus, strategies, such as use of immunosuppressive agents
to suppress immune reaction have been proposed. However, this is
not only difficult to achieve, but may not be beneficial to the
ultimate outcome of the therapy, since the immune system cannot
then perform its function such as fighting infection, other tumor
cells and pathogens.
Accordingly, it would be desirable to have a nucleic acid delivery
agent that can be assembled more simply than other nucleic acid
delivery systems, such as the delivery system of Wu and Wu.
It would also be desirable if such a delivery system could be
synthesized more readily than is possible with a chemical coupling
process.
It would also be desirable that the nucleic acid delivery system
could readily be adapted to be used to specifically target a
variety of target cells
It would also be desirable if the delivery system had lower
antigenicity than many currently available delivery systems. For
example, it would be desired if it could be used to deliver a
cytotoxic agent, e.g. an immunotoxin, to a cell without the
antigenicity currently associated with such systems.
It would also be beneficial if this system did not have the
potential of causing disease on its own by malignant transformation
of a cell such as can occur with viral delivery systems.
SUMMARY OF INVENTION
We have now developed a highly efficient nucleic acid delivery
system to a desired target cell. This system can be used, for
example, to deliver a gene coding for the essential portion of a
toxin protein. The nucleic acid, either DNA or RNA, is coupled to a
fusion protein. The fusion protein consists of a target moiety and
a nucleic acid binding moiety, for example a DNA binding moiety.
For example, the target moiety preferably can be an antibody, more
preferably a single chain antibody, a Fab portion of the antibody
or a (Fab').sub.2 segment. If the target animal is a human, the DNA
binding moiety should preferably be a human DNA binding moiety,
such as protamine.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of one embodiment of the use
of nucleic acid delivery system according to the present
invention.
FIG. 2 is a schematic representation of one embodiment of the
expression vector for the fusion protein. It is a schematic
representation of a bi-cistronic mammalian expression vector, which
will encode an antibody for the HIV gp120 protein fused to a
protamine protein.
FIG. 3 is an autoradiograph showing radiolabeling and
immunoprecipitation of expressed Fab105-protamine fusion
proteins.
FIG. 4 shows purification and SDS-PAGE analysis of the recombinant
fusion proteins.
FIG. 5 shows binding activity of the purified fusion proteins to
HIV gp120.
FIG. 6 is an autoradiograph showing the DNA binding activity of the
Fab105-protamine fusion proteins under varying concentrations.
FIG. 7 is an autoradiograph showing the DNA binding activity of the
Fab105-protamine fusion proteins under varying concentrations.
FIG. 8 are FACS analysis showing the binding ability of
Fab105-protamine DNA complexes to gp120 protein as compared to that
of Fab105 complexes in both uninfected and HIV-infected cells.
FIG. 9 is a schematic of the expression vectors of the PEA
catalytic fragment schematically showing the PEA encoding gene and
two vectors made containing partial domains of this gene.
FIG. 10 is a graph showing selective cytotoxicity of one of the
nucleic acid delivery systems of the present invention,
Fab105-protamine-toxin expressor, to HIV infected cells and shows
cell viability.
FIG. 11 is a graph showing selective cytotoxicity of
Fab105-protamine-toxin expresser complexes to HIV-infected cells
and shows a protein inhibition assay.
FIG. 12 shows selective cytotoxicity of the Fab105-protamine-toxin
expressor complexes to HIV-infected cell as measured by
ADP-ribosylation activity.
FIG. 13 is a reproduction of FIG. 1 of Talanian, et al., Science
249:769 771 (1990).
FIG. 14 is a reproduction of FIG. 1A of Ashley, et al., Science
262:563 566.
FIG. 15 is a reproduction of FIG. 1B of Ashley, et al., supra.
FIG. 16 is a reproduction of FIG. 1 of Rabindran, S. K., et al.,
Science 259:230 234 (1993).
DETAILED DESCRIPTION OF THE INVENTION
We disclose herein a new nucleic acid delivery system. The system
comprises a fusion protein which binds the desired nucleic acid
sequence.
The fusion protein comprises a target moiety and a binding moiety.
The target moiety is preferably a protein that will specifically
bind to a site on the target cell. For example, it can be a ligand
for a ligand specific receptor for instance a fibroblast growth
factor receptor (FGF-R)) and the specific FGF for that receptor,
e.g. basic FGF for a basic FGR-R. Alternatively, the protein can be
an antibody specific to the target cells. For example, it can be an
antibody to an HIV envelope protein, an antibody to an oncogenic
determinant such as extracellular ligand-binding domain of an
activated receptor, (e.g., erbB, kit, fms, neu ErbB2), etc. It can
also be an antibody to a receptor, for example, an antibody to the
GM-CSF receptor. Preferably, the target moiety is an antibody.
Still more preferably, the antibody is a single chain antibody
comprising the binding sequence of the antibody, a (Fab').sub.2
segment or the Fab fragment of the antibody. More preferably the
antibody is a single chain antibody or a (Fab').sub.2 segment.
The particular target moiety chosen can be determined empirically
based upon the present disclosure depending upon the target cell.
For example, with somatic cell therapy or in vivo with readily
accessible cells or tissues such as an intravascular target, the
important attributes of the target moiety are affinity and
selectivity. In such instances the use of single chain antibodies
as the target moiety is preferable. However, when the target cell
is not readily accessible, such as when the cell is part of a large
solid tumor mass with a poor blood supply and high interstitial
pressure, the serum half--life is extremely important to consider.
In such instances, the full antibody and (Fab').sub.2 segments are
typically preferred. In a preferred embodiment, one could
synthesize the fusion protein so that the binding moiety is
attached to the carboxy terminus of an intact immunoglobulin such
as IgG.sub.1.
In order to limit antigenic reaction, the targeting moiety is
preferably selected to take into account the host animal whose
cells will be targeted. Thus, if the target animal is a mouse, one
preferably uses murine antibodies, whereas if the target animal is
a human, one preferably uses a human antibody or a humanized
antibody.
The second part of the fusion protein consists of a nucleic acid
binding moiety, either a DNA or RNA binding moiety. Preferably, one
uses a moiety that can bind either DNA or RNA. This binding moiety
can be any protein from the target animal that will bind either DNA
or RNA. For example, it can be protamine, which is a small basic
DNA binding protein, which serves to condense the animal's genomic
DNA for packaging into the restrictive volume of a sperm head
[Warrant, R. W., et al., Nature 271:130 135 (1978); Krawetz, S. A.,
et al., Genomics 5:639 645 (1989)]. The positive charges of the
protamine can strongly interact with negative charges of the
phosphate backbone of nucleic acid, such as DNA resulting in a
neutral and stable DNA--protamine complex. The nucleic acid can be
either DNA or RNA depending on the purpose. For example, the
nucleic acid to be transferred can be used to express an antibody
intracellularly, dominant negative mutants, anti-sense RNA,
ribozymes or a cytotoxic agent. For example, the cytotoxic agent
can be a portion of a bacteria or plant toxin which is extremely
potent such as ricin, the catalytic fragment of Pseudomonas
exotoxin A (PEA), etc.
The nucleic acid can be used for transient or table transfection of
the cell. For example, when the nucleic acid encodes a factor which
is lethal to the cell such as a DNA segment encoding a toxin,
transient expression is sufficient. In contrast, where it expresses
a factor such as a suppressor gene (e.g. retinoblastoma), or a
protein that is not being expressed at sufficient levels, e.g.
adenosine deaminase (ADA) [Belmont, J. W., et al., Mol. & Cell.
Biol. 8:5116 5125 (1988); Palmer, T. D., et al., Proc. Natl. Acad.
Sci USA:1055 1059 (1987), uridine diphosphate
(UDP)-glucuronyl-transferase [Ponder, K. P., et al., Proc. Natl.
Acad. Sci USA 88:1217 1221 (1991)], or insulin, stable integration
into the cells chromosome may be desired. In those instances where
stable integration is desired the nucleic acid can be a DNA segment
wherein the gene coding for the desired factor is inserted into a
cassette that will facilitate integration into the cell. For
instance, the integration cassette which surrounds the gene can be
a 5' and 3' LTR (long terminal repeat) of a retrovirus i.e. MMLV,
an ITR (inverted terminal repeat unit, i.e. adeno associated
virus), etc. [See, e.g., Scherdin, U., et al., J. Virol, 64:907 912
(1990); Stief, A., et al., Nature 341:343 345 (1989); Phi-Van, L.,
et al., Mol. & Cell. Biol. 10:2302 2307 (1990); Phi-Van, L. et
al., The EMBO Journal 7:655 664 (1988)]. This cassette can be
prepared by standard techniques. For example, mammalian expression
vectors where a gene of interest can be inserted between LTRs or
ITRS. One can construct a cassette containing flanking LTR or ITR
regions at both ends, a promoter/enhancer, preferably with a
polylinker for the gene of interest to be inserted in between, and
when desired a selectable marker based upon the present disclosure
using known techniques. This cassette with the desired nucleic
acid, e.g. gene or genes, of interest is the nucleic acid
segment.
The target moiety specifically brings the delivery system to the
target cell.
One can also use localization sequences to intracellularly deliver
the released RNA or DNA to a cellular site of interest.
Thereafter, the targeted cell can internalize the delivery system,
which is bound to the cell. Typically, the delivery system binds to
a specific receptor on the cell.
For example, membrane proteins on the cell surface, including
receptors and antigens can be internalized by receptor mediated
endocytosis after interaction with the ligand to the receptor or
antibodies. [Dautry-Varsat, A., et al., Sci. Am. 250:52 58 (1984)].
This endocytic process is exploited by the present delivery system.
Because this process can damage the DNA or RNA as it is being
internalized, it is preferable to include a strong promoter for the
nucleic acid that is to be expressed. Similarly, the use of a
segment containing multiple repeats of the gene of interest may be
desirable. One can also include sequences or moieties that disrupt
endosomes and lysosomes. see, e.g., Cristiano, R. J., et al., Proc.
Natl. Acad. Sci. USA 90:11548 11552 (1993); Wagner, E., et al.,
Proc. Natl. Acad. Sci. USA 89:6099 6103 (1992); Cotten, M., et al.,
Proc. Natl. Acad. Sci. USA 89:6094 6098 (1992).
In deciding what type of nucleic acid segment to use, the skilled
artisan will take into account the protein being expressed in light
of the present specification. For example, when one is introducing
a toxin protein, because of its extreme cytotoxicity, the
expression of only a few molecules are needed to kill a cell. In
other cases such as with expressing ADA, larger amounts of protein
expression are needed and the use of LTRs, ITRs, as part of the DNA
cassette, and/or lysosomal disrupting agents such as
replication-defective adenoviruses may be used.
The particular protein chosen for the targeting moiety will depend
upon the target cell. For example, if one is targeting an infected
cell, such as an HIV infected cell, one can use a monoclonal
antibody that will specifically target HIV infected cells. This
would include an antibody against the envelope glycoprotein. One
can use any of a number of known antibodies against HIV-1 gp120 or
HIV-2 gp120, such as 15e, 21h [Thali, M., et al., J. Virol. 67:3978
3988 (1993)], F105, 176 and 48d. If one wants to deliver the
nucleic acid sequence prophylactically such as a gene for
intracellular expression of an antibody, a decoy sequence, etc.,
one can target highly susceptible cells by targeting receptors
present on such cells such as the CD4 receptor for HIV susceptible
cells. In such a situation, the protein can be a ligand that will
preferentially bind to the receptor, for example, CD4, as well as
using an antibody to the receptor, such as an antibody to the CD4
receptor.
This strategy for choosing the targeting moiety is very adaptable.
For example, certain tumors are frequently associated with cells
possessing a large amount of a particular cell surface receptor
(e.g. neu with breast cancers), or an abnormal form of a particular
protein.
Other receptors of interest include those for lymphokines such as
interleukins and interferons, for example, the interleukin-2 (IL-2)
receptor (IL-2R). The p55, IL-2R .alpha. chain also referred to as
the Tac protein is associated with Ag or mitogen-activated T-cells
but not resting T-cells. It is expressed in high levels on
malignant cells of lymphoid cancers such as adult T-cell leukemia,
cutaneous T-cell lymphoma and Hodgkins disease. The anti-Tac
antibody will bind to this protein. Humanized version of such
antibodies are known and described in Queen, C., et al., Proc.
Natl. Acad. Sci. USA:10029 10039 (1989); Hakimi, J., et al., J. of
Immun. 151:1075 1085 (1993) (Mik.beta.1 which is a Mab against
IL-2R .beta. chain); Kreitman, R. J., et al., J. of Immun. 149:2810
2815 (1992); Hakimi, J., et al., J. of Immun. 147:1352 1359
(1991).
Antibodies to these various proteins are known and available. These
antibodies can readily be adapted for use in this system by
following the general procedures described herein, and substituting
the gene coding for the desired binding site for the exemplified
gene. For example, where the targeted cell is an HIV-infected cell,
the targeting moiety can target the HIV envelope glycoprotein. Any
number of antibodies to this protein can be used. For instance, a
recombinant antibody based on the F105 antibody is made by known
teachings techniques. [Posner, M. R., et al., J. Immunol. 146:4325
4332 (1991); Thali, M., et al, J. Virol. 65:6188 6193 (1991);
Marasco, W. A., et al., Proc. Natl. Acad, Sci. USA 90:7889 7893
(1993)] other antibodies that can be made include, 15e, 21h, 17b,
48d, etc.
A vector for expression of the antibody can be made as described
herein. For example, a bicistronic mammalian expression vector
which will express the Fd portion of the antibody (V.sub.H and
C.sub.H) and the binding region of the light chain (e.g. a kappa
chain) of, for example, the F105 antibody can be constructed by
using an Fd fragment without a stop codon and amplifying the
segment by standard techniques, for example by polymerase chain
reaction (PCR). The upstream primer preferably wil correspond to
the leader sequence of the immunoglobulin of the animal from which
the cells of the delivery agent is to be used (for example, where
the target cell is a human cell a human immunoglobulin of amino
acids 1 6), with an additional convenient cloning site such as a
HindIII site. The downstream primer can correspond to amino acids
by the carboxy terminus of the heavy chain constant region. For
example, with an antibody based upon F105, amino acids 226 233 of
human heavy chain CHI domain with a convenient cloning site
inserted, such as the XbaI site. The PCR reaction is performed
according to standard means. By this means the gene or gene segment
encoding the targeting moiety of the fusion protein is
prepared.
As described above, the second portion of the fusion protein is the
binding moiety. Preferably, one uses a single vector containing
gene segments that will express both the targeting moiety and the
binding moiety. However, one can use a vector system to
co-transfect a cell with at least two vectors and select for cells
expressing the fusion protein. Preferably, one uses a single
vector. One preferably attaches the sequence encoding the target
moiety to a gene, or gene segment, encoding the binding moiety by
standard means. For example, a gene for human protamine [Balhorn,
J. of Cell. Biol. 93:298 305 (1982)]. Other nucleic acid binding
proteins include GCN4, Fos and Jun which bind DNA through a common
structural motif consisting of several basic residues and an
adjacent region of about 30 residues containing a heptad repeat of
leucines, the "leucine zipper" that mediates dimerization
[Talanian, R. V., et al., Science 249:769 771 (1990) Talanian, et
al. state at 769: This "bZIP" (4) [4. C. R. Vinson, P. B. Sigler,
S. L. McKnight, Science 246, 911 (1989)] motif consists of a region
with several basic residues that probably contacts DNA directly and
an adjacent region of about 30 residues containing a heptad repeat
of leucines, the "leucine zipper" (5) [5. W. H. Landschultz, P. B.
Sigler, S. L. McKnight, ibid. 240, 911 (1989)], that mediates
dimerization. Such bZIP dimers bind DNA sites that are
approximately diad-symmetric (3) [Reviewed by P. F. Johnson and S.
L. McKnight, Annu. Rev. Biochem. 58, 799 (1989); K. Struhl, Trends
Biochem. Sci. 14, 137 (1989)]. and later: A peptide (GCN4-brl),
corresponding to residues 222 to 252 of GCN4 (22) [22. G. Thireos,
M. D. Penn, H. Greer, Proc. Natl. Acad. Sci. U.S.A., 81, 5097
(1984); A. G. Hinnebusch, ibid., p. 6442.], was synthesized (23)
[Peptides were synthesized on an Applied biosystems Model 430A
peptide synthesizer with standard reaction cycles modified to
include acetic anhydride capping. Peptides were cleaved from the
resins by low-high HF cleavage (Immunodynamics, Inc., San Diego,
Calif.) and desalted by Sephadex G-10 chromatography in 5% acetic
acid. Purifications were by high-performance liquid chormatography
with a Vydac reversephase C.sub.18 column and a linear gradient of
CH.sub.3CN--H.sub.2O with 0.1% trifluoroacetic acid. Fast atom
bombardment mass spectrometry; GCN4-brl: calculated, 3796.5; found,
3795.8; GCN4-bZIPl: calculated, 7015.4 found, 7015.5.]with a
Gly-Gly-Cys linker (6) [6. E. K. O'Shea, R. Rutkowski, P. S. Kim,
ibid. 243, 538 (1989)] added at the carboxyl terminus (FIG. 1). The
glycines were included to provide a flexible linker in the
disulfide-bonded dimer, referred to as GCN4-brl.sup.ss''. The
peptide was made as the carboxylterminal amide to avoid
introduction of additional charge. A second peptide (GCN4-bZIPl),
corresponding to the entire bZIP region of GCN4 (residues 222 to
281), was also synthesized (FIG. 1). This 60-residue peptide is
capable of dimerization and sequence-specific DNA binding (8) [8.
I. A. Hope and K. Struhl, Cell 46, 885 (1986)]. FIG. 1 at 769
states: Sequences of the peptides studied (23) [23. Peptides were
synthesized on an Applied biosystems Model 430A peptide synthesizer
with standard reaction cycles modified to include acetic anhydride
capping. Peptides were cleaved from the resins by low-high HF
cleavage (Immunodynamics, Inc., San Diego, Calif.) and desalted by
Sephadex G-10 chromatography in 5% acetic acid. Purifications were
by high-performance liquid chormatography with a Vydac
reverse-phase C.sub.18 column and a linear gradient of
CH.sub.3CN--H.sub.2O with 0.1% trifluoroacetic acid. Fast atom
bombardment mass spectrometry; GCN4-brl: calculated, 3796.5; found,
3795.8; GCN4-bZIPl: calculated, 7015.4 found, 7015.5.]. GCN4-bZIPl
consists of the 60 carboxyl-terminal residues of GCN4 (22). The
leucines in the leucine repeat are underlined. GCN4-brl consists of
the basic region residues (boxed) plus the carboxyl-terminal linker
Gly-Gly-Cys. Abbreviations for the amino acid residues are: A, Ala;
C, Cys; D, Asp; E, Glu; G, Gly; H, His; K, Lys; L, Leu; M, Met; N,
Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.];
the TFIIS nucleic acid binding domain, which is seen in the
C-terminal residues 231 280 [Qlan, X., et al., Nature 365:277 279
(1993)]; the ribonucleoprotein (RNP) family that is present in
domains in human FMRI, the yeast protein HX, 14 domains of the
chicken gene vigillin, mer1, a yeast protein, bacterial
polynucleotide phosphoylase, and the ribosomal protein S3 [Ashley,
C. T., et al., Science 262:563 566] Ashley, et al. state in FIG. 1
at 563: Location and homologies of RNP family domains in FMRP.
[FIG. 14] (A) Alignment (27) [27. M. Gribskov, R. Leuthy, D.
Eisenberg, Methods Enzymol. 183, 146 (1989)]. The 12-residue
element is not found in the sequence of Drosophilia HSF. It is
possible that another element may serve the same function.] of the
amino acid sequences that make up the KH domains of FMRP and
several other proteins and the corresponding consensus sequence.
Numbers in parentheses indicate the particular domain shown for the
proteins that have multiple KH domains, and the number preceding
the first residue indicates that position in the corresponding
protein. Dark highlighting indicates similarities among all
proteins, whereas stippled highlighting indicates similarity
between the two KH domains of FMRP. Boldface residues show the
positions of polar amino acids, indicated by if in the consensus
sequence. The bracketed lysine (K) residues indicate this amino
acid at either position in the domain. The position of the
isoleucine-to-asparagine mutation at position 304
(I.sup.304.fwdarw.N) in a patient (6) [D. Wohrle, et al., Am. J.
Hum Genet. 51, 299 (1992); A. K. Gideon, et al.. Nature Genet. 1,
341 (1992); K. De Boulle et al., ibid. 3, 31 (1993)] is indicated
at the bottom. [FIG. 15] [1. W. T. Brown, Am. J. Hum. Genet. 47,
175 (1990); S. L. Sherman et al., Ann. Hum. Genet. 48, 21 (1984);
2. M. G. Butler, T. Mangrum, R. Gupta, D. N. Singh, Clin. Genet.
39, 347 (1991); 3. A. M. J. G. Verkerk, et al., Cell 65, 905
(1991); I. Oberle et al., Science 252, 1097 (1991); E. J. Kremer et
al., ibid. p. 1711; A. Vincent et al, Nature 349, 624 (1991)]. (B)
Diagram of FMRP [residue numbers are as described (7)]. [7. C. T.
Ashley et al., Nature Genet. 4, 244 (1993)] The CGG repeat and
initiating codon (M.sup.1) are indicated as is each KH domain,
labeled 1 and 2. Also shown is the amino acid sequence with the two
RGG box domains highlighted. Abbreviations for the amino acid
residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,
His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S. Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. FIGS. 1a and b are
reproduced as FIGS. 14 and 15.; and the binding motifs in heat
shock protein [Rabindran, S. K., et al., Science 259:230 234
(1993)] Rabitindran, et al., state in FIG. 1 at 231: FIG. 1. [FIG.
16] Activity of wild-type and mutant human HSF1 proteins
transiently expressed in 293 cells. Map of wild-type and mutant
human HSF1 (hHSF) ORFs is at left. Numbers on the right indicate
the end point of the truncated fragments; amino acids in the fourth
hydrophobic repeat and appended by cloning at the COOH-terminal and
are represented by the single-letter code (30). [30. Abbreviations
for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp;
E, Glu; F, Phe; G, Glyp; H, His; I, Ile; K, Lys; L, Leu; M, Met; N,
Asn; P, Pro; Q,. The host animal of the target cells will be used
to determine which protein or protein fragment with a binding motif
is used. For example, with a human host and for expression of human
protamine one can use the known plasmid pTZ 19R-HP1 [Krawetz, S.
A., et al. Genomnics 5:639 645 (1989)]. Preferably one would delete
an intron in this gene so that the expression vector can also be
used for expression of the fusion protein in prokaryotic systems as
well as eukaryotic systems. PCR amplification would be performed by
standard means. For example, using an upstream primer, which
corresponds to sequences from the amino terminus, for example,
corresponding to amino acids 1 6 of the protamine protein with a
convenient restriction site, such as the XbaI cloning site and a
downstream primer corresponding to the carboxy portion of the first
exon, for example, amino acids 29 37 with additional sequences
complimentary to the 5' amino acids in the second terminus (e.g.
amino acids 38 40 in the second exon). A second PCR reaction can
then be performed using the upstream primer corresponding to the
amino terminus and the downstream primer corresponding to an
overlapping portion to the carboxy terminus. For example, using a
sequence corresponding to amino acids 31 40 with the sequence of
amino acids 41 to the stop codon in the second exon and an
additional convenient cloning site such as NotI. The first PCR
amplified DNA segment can be used as a template. By using
convenient restriction sites, one can cut out the targeting moiety
and the binding moiety by known methods such as purifying them
using standard techniques, e.g., agarose gel, For example, in the
example described above, the PCR amplified Fd of F105 without a
stop codon can be cut with HindIII/XbaI and purified by agarose
gel. The PCR amplified protamine coding gene, without intron can be
cut with XbaI/NotI and purified from agarose gel. The Fab105
plasmid can be cut with HindIII/NotI and the DNA segment purified
from an agarose gel The HindIII/XbaI-cut Fd fragment and
XbaI/NotI-cut protamine fragment can then be cloned into the
HindIII/NotI sites of the plasmid containing F105 by three-piece
ligation. See FIG. 2. The resulting expression vector thus contains
a cartridge of an Fd-protamine fusion gene (in-frame) and
kappa-chain gene under the control of an independent promoter, such
as a CMV promoter. The particular promoter that will be used
depends upon the desired cell system for expression of the fusion
protein. Promoters are known to the skilled artisan and can readily
be selected based upon the present disclosure. For example,
preferred promoters include CMV, SR.alpha., RSV, MMLV LTR, SV40 and
HIV-1 5' LTR.
This construct can readily be confirmed by standard means, such as
DNA sequencing.
This expression vector can then be used to stably transform a cell
line. The cell line can be any desired cell line including
prokaryotic as well as eukaryotic cells. Preferred cell lines
include mammalian cell including COS cells, kidney cell lines such
as CHO, myeloma cell lines such as SP/0, and SP/2, HMMA2 11 TG10,
and insect cell lines such as Drosophilla. Preferably, to reduce
antigenicity one would use a mammalian cell line. More preferably,
one would use a myeloma cell. Preferred cells include SP/0, SP/2,
Sp2/0-Ag14, X63Ag8.653, FO, NSI/1-Ag4 1, NSO/1, FOX-NY, YB2/0 and
1R983F.
The transformation of the cell can be by any standard techniques.
It is preferred that one stably transforms the cell, although in
certain instances transient transformation by the DEAE-Dextran
technique will be acceptable. Thus, one preferably uses a method
for stably transforming the cell, such as the calcium phosphate
precipitation method followed by selection of transformed cell
lines such as by G418 selection. The transformed cell line can be
cultured and the fusion protein harvested by standard techniques.
For instance, the Fd-protamine protein and kappa-chain of F105 are
expressed and secreted into the culture of COS transformed Fab105
protamine cells and detected by radiolabelling and
immunoprecipitation with anti-human IgG antibody.
For example, a COST cell can be transfected with an expression
vector containing the cartridge of the targeting moiety-binding
moiety using lipofectin. Vectors include the vector
pCMV-Fab105-protamine. The transfected cells can then be incubated
in DMEM, supplemented with 10% fetal calf serum (FCS) for two days
and replaced with a selection medium such as DMEM with 10% FCS and
500 .mu.g G418. This is readily available, for example, from BRL.
The G418 resistant colonies will appear after about two weeks and
can be readily selected. Colonies can then be cloned with limiting
dilution and examined by radiolabelling and immunoprecipitation,
ELISA and immunofluorescent staining for expression of the
recombinant fusion protein. The proteins can be secreted and
purified in these cells by standard means. For example, the
transformed COS cells can be grown in a flask with DMEM medium
supplemented with 10% fetal calf serum and 500 .mu.g/ml of
neomycin. After reaching confluence, the cultures can be replaced
with fresh DMEM without FCS every three days for two weeks. The
collected culture mediums can be clarified by, for example,
centrifugation at, for instance, at 500 rpm for 20 minutes at
4.degree. C. and then concentrated using, for example, a membrane
filter with a molecular weight cutoff of 10,000 dalton such as an
Amico concentrator. The concentrated medium can then be loaded into
an affinity column coupled with anti-human IgG kappa-chain
monoclonal antibody, such as sold by Kirkegaard & Perry, Inc.
The affinity column can be washed with PBS and loaded with the
concentrated culture medium. The medium will then pass through the
column, followed by PBS washings until no protein is detected in
the eluate. The column is then washed with pre-elution buffer, for
example, 10 mM phosphate at pH 8.0 and eluted from the column with
100 mN glycine at pH 2.4. The protein peak fractions are detected
by standard means such as by for example Bradford protein assay
(Biolab) and pooled together and dialyzed against 0.2 M NaCl.
In a preferred embodiment you would adapt the cell for growth in a
serum-free medium. This can be done by the skilled artisan. For
example, one can readily adapt COS and CHO cells. In doing this,
relatively pure Fab-fusion proteins are secreted into the medium.
Thus, the purification procedure is simpler.
The purified fusion protein is now ready to be combined with the
desired nucleic acid sequence such as one for a positive
potentiator (such as a gene for a cytokine, a gene for a missing or
detective protein, etc.) or a sequence for a negative potentiator
(such as a toxin, an anti-sense RNA, a suicide gene such as HSV
thymidiac kinase, a ribozyme, a dominant-negative mutant, etc.).
For example, when the nucleic acid encodes a toxin, one preferably
takes care to alter the toxin gene to minimize its potential to
affect non-targeted cells. This can be done by standard techniques
such as deleting those sequences encoding recognition domains.
Toxins are well known and include diphtheria toxin and truncated
versions thereof, pseudononas exotoxin, and truncated versions
thereof, Ricin/abrin, Blocked ricin/abrin, Ricin ToxinA-chain,
ribosome inactivating protein, etc. All these proteins have
different domains. For example, the gene encoding PEA has several
domains: Domain I is responsible for cell recognition; Domain II
for translocation of the toxin cross-membrane and Domain III for
adenosine diphosphate (ADP)-ribosylation of elongation factor 2,
which is the step actually responsible for cell death. [Gary, G.
L., et al., Proc. Natl. Acad. Sci. USA 81:2645 2649 (1984);
Allured, V. S., et al., Proc. Natl. Acad. Sci. USA 83:13220 1324
(1986); Siegall, C. B., et al., J. Biol. Chem. 264:14256 14261
(1989)]. Accordingly, by alterations in Domain I or Domain II, that
render those domains incapable of expression, for example, by a
frameshift mutation, insertion of termination sequences, or
deletions one can minimize the ability of the toxin to affect
neighboring cells. Thereafter, the skilled artisan can use standard
techniques to insure that the other domains, or portions of domains
where expression is desired, are used.
For example, as indicated above, with PEA only Domain III is
absolutely required. However, we have found that including partial
sequences from other domains makes the toxin more effective. For
example, we prepared two PEA mammalian expression vectors. This is
one in which Domain III (mature PEA amino acid residues 405 to 613)
only, referred to as pCMV-PEA III is expressed and one which
encodes Domain III and partial Domain IB, a sequence of amino acids
385 to 613 (pcMV-PEAIbIII) is expressed. These sequences should be
operably linked to a promoter which will permit expression in the
target cell. For example, mammalian promoters such as CMV,
SR.alpha., RSV, SV40, MMLV LTR, HIV-1 5' LTR, are preferred. More
preferably, CMV, HIV-1 5' LTR, RSV, and SV40. The toxin proteins
encoded by these gene fragments lack a recognition domain. They are
non-toxic to surrounding cells and are only toxic when expressed
inside a cell. These expression vectors can readily be tested to
determine how well they express a product intracellularly by a
simple in vitro assay. For example, the expression of those DNA
sequences encoding PEA toxin fragments can be tested by
transforming a cell with the delivery system and observing the
cytotoxicity of the cell. We have found that the pCMV-PEIbIII
vector shows a higher level of ADP-ribosylation than the vector
expression only Domain III and thus, we prefer using it.
FIG. 1 is a schematic representation showing the use of a nucleic
acid delivery system according to the present system, wherein the
nucleic acid sequence is a toxin expressor DNA.
In some instances, even with immunotoxins, resistant mutants can
develop. In such instances, one can readily insert a different
toxin gene or different types of nucleic acid segments into the
nucleic acid cassette which is attached to the fusion protein.
Thus, the present system permits the production and use of a wide
range of DNA and RNA segments.
In some preferred embodiments one would administer a cocktail of
nucleic acid delivery systems where the targeting moiety may be
changed to broaden the number of targeted cells or alternatively
the nucleic acid segment that is delivered is changed to widen the
spectrum of products delivered to the target cell.
When the protein is a toxin, transient expression in the cell is
all that is needed. However, when it is desired to stably transform
a cell, the gene is placed into a cassette containing LTRs or ITRs
at either side to foster stable integration. Alternatively the
cassette can be an episomal vector such as one that contains an
Epstein Barr virus for example, pEBV His A, B, and C, pREP4, pREP7,
pREP10, which are sold commercially by Invitrogen Corporation.
The recombinant fusion proteins are combined with the nucleic acid
segment by standard techniques. For example, the fusion protein can
be mixed with given amounts of the desired nucleic acid sequence,
either DNA or RNA, by known means such as mixing in solution. For
example, in 0.2 M NaCl solution. The DNA or RNA is readily bound by
the protamine. Thereafter, the carrier can be administered to the
desired cell either for somatic cell therapy or used in vivo.
The delivery system can be delivered by any of a number of means.
For example, it can be administered by parenteral injection
(intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.)
or subcutaneous (s.c.)), oral or other routes of administration
well known in the art. Parenteral administration is preferred.
The amount used will typically be in the range of about 0.1 mg to
about 10 mg/kg of body weight. The delivery system will preferably
be formulated in a unit dosage form based upon the nucleic acid or
nucleic acids being delivered.
For example, solid dose forms that can be used for oral
administration include capsules, tablets, pills, powders and
granules. In such solid dose forms, the active ingredient, i.e.,
targeting moiety, is mixed with at least one inert carrier such as
sucrose, lactose or starch. such dose forms can also comprise
additional substances other than inert diluents, e.g., lubricating
agents, such as magnesium stearate. Furthermore, the dose forms in
the case of capsules, tablets and pills may also comprise buffering
agents. The tablets, capsules and pills can also contain
time-release coatings.
For parenteral administration, one typically includes sterile
aqueous or non-aqueous solutions, suspensions or emulsions in
association with a pharmaceutically acceptable parenteral vehicle.
Examples of non-aqueous solvents or vehicles are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil and corn oil,
gelatin and injectable organic esters, such as ethyl oleate. These
dose forms may also contain adjuvants such as preserving, wetting,
emulsifying and dispersing agents. They may be sterilized by, for
example, filtration through a bacterial-retaining filter, by
incorporating sterilizing agents into the composition, by
irradiating the compositions, etc., so long as care is taken not to
inactivate the antibody. They can also be manufactured in a medium
of sterile water or some other sterile injectable medium before
use. Further examples of these vehicles include saline, Ringer's
solution, dextrose solution and 5% human serum albumin. Liposomes
may also be used as carriers. Additives, such as substances that
enhance isotonicity and chemical stability, e.g., buffers and
preservatives, may also be used.
The preferred range of active ingredient in such vehicles is in
concentrations of about 1 mg/ml to about 10 mg/ml. More preferably,
about 3 mg/ml to about 10 mg/ml.
Although receptor mediated gene delivery in certain embodiments may
be relatively inefficient, by utilizing the present gene delivery
system, one does not have to worry about antigenic reactions
occurring from the use of higher dosages or repeated injections.
This is because the targeting moiety and the DNA binding moiety can
be designed so that they are either from the animal that one is
injecting, such as a human, or made to be like that animal, i.e.
using a humanized murine antibody or binding protein for a human.
DNA itself is weakly or non-immunogenic. Thus, the entire agent is
either non or weakly immunogenic. Since the delivery system can be
efficiently produced and adapted to have high binding activity, it
can be used repeatedly.
Additionally, as discussed above there are methods that can be
utilized in the present system to improve the efficiency of the
delivery system. For example, one can include targeting sequences
such as nuclear targeting sequences associated with the nucleic
acid segment to more efficiently deliver the nucleic acid to its
desired target. Targeting sequences are known in the art and
include for example, the nuclear localization signal on HIV gag p17
between positions 25 and 33 (SEQ ID NO:1) (KKKYYKLK). Thus, one can
include a targeting sequence, preferably inside a liposome, as part
of the fusion moiety to more effectively target the DNA.
The present invention is further illustrated by the following
examples. These examples are provided to aid in the understanding
of the invention and are not to be construed as a limitation
thereof.
EXAMPLES
A bi-cistronic mammalian cell expression vector
(pCMV-Fab105-Protamine) which contains a chimeric gene encoding the
Fd of F105 fused to the human protamine protein in one expression
cassette and the F105 kappa chain encoding gene in another
expression cassette (FIG. 2) was constructed as follows.
Construction of Mammalian Expression Vector For Fab105-Protamine
Fusion Protein
The pCMV-Fab105 plasmid was constructed as described below. This
plasmid contains bi-cistronic expression cassettes for the Fd gene
and kappa chain gene derived from F105 hybridonma. To construct a
fusion protein expression vector, the Fd fragment of F105 without a
stop codon was amplified by PCR using the pCMV-Fab105 as a
template. The upstream primer (SEQ ID NO:2)
(5'-TTTGAATTCAAGCTTACCATGGAACATCTGTGGTTC-3') corresponding to the
leader sequence of human immunoglobulin of amino acids 1 to 6 with
an additional HindIII cloning site (Kabat, et al., 1987), and the
downstream primer (SEQ ID NO:3)
(5'-GGTACCGAATTCTCTAGAACAAGATTTGGGCTC-3') corresponding to the
amino acids of 226 to 233 of human heavy chain constant region with
an additional XbaI cloning site were used for PCR amplification.
The PCR reaction was performed as described previously [Marasco, et
al., J. of Clin. Invest. 90:1467 1478 (1992)].
The human protamine gene was amplified from the plasmid pTZ19R-HP1
[Krawetz, S. A., et al., Genomics 5:639 645 (1989)]. To delete an
intron in the protamine gene in this clone (for expression in
prokaryotic system as well), the first PCR amplification was
performed using the upstream primer P1, (SEQ ID NO:4)
(5'-GGTACCGAATTCTCTAGAATGGCCAGGTACAGATGC-3') which corresponds to
the sequence of amino acids 1 to 6 of protamine protein with an
additional XbaI cloning site, and the downstream primer (SEQ ID
NO:5) (5'-TTTAGGATCCTTAACAACACCTCATGGCTCTCCTCCGTGTCTGGCAGC-3')
which corresponds to amino acids 29 to 37 with additional sequence
complementary to amino acids 38 to 40 in the second exon. The
second PCR reaction was performed using the upstream primer P1 and
the downstream primer, (SEQ ID NO:6)
(5'-TTAATTGCGGCCGCTTAGTGTCTTCTACATCTCGGTCTGTACCTGGCGCTGACACCTCATGGCTCTCCT-
CCGTGTCTG3'), corresponding to amino acid sequence 31 to 40 with
the sequence of amino acids 41 to stop codon in the second exon and
an additional NotI cloning site. The first PCR-amplified DNA was
used as a template.
To construct a bi-cistronic fusion protein expression vector, the
PCR-amplified Fd of F105 without a stop codon was cut with
HindIII/XbaI and purified from an agarose gel, The PCR-amplified
entire protamine coding gene without intron was cut with XbaI/NotI
and purified from an agarose gel. The pCMV-Fab105 plasmid was cut
with HindIII/NotI and the DNA fragment about 7.0 kd was purified
from an agarose gel. The HindIII/XbaI-cut Fd fragment and
Xbal/NotI-cut protamine fragment were then cloned into the
HindIII/NotI sites of pCMV-Fab105 by three-piece ligation. The
resultant expression vector, designated as pCMV-Fab105-Protamine,
contains Fd-protamine fusion gene (in-frame) and kappa chain gene
under the control of independent CMV promoter. This construct was
confirmed by DNA sequencing.
The F105 Fd and human protamine DNA fragments which were cloned
into the pCMV-Fab105 vector are shown in FIG. 2. The resulting
bi-cistronic expression vector (pCMV-Fab105-Protamine) contains an
expression cassette for the Fd105-Protamine fusion protein and
another cassette for the kappa chain of F105.
A transformed mammalian cell line COS-Fab105-Protamine was
generated after DNA transfection and G418 selection.
Construction of Transformed Cell Lines
To generate transformed cell lines, COS-1 cells were grown on
6-well plates and transfected with pCMV-Fab105-Protamine using
lipofectin as described previously [Chen, S, -Y., et al., J. Virol.
65:5902 5909 (1991)]. The transfected cells were incubated in DMEM
supplemented with 10% FCS for two days and replaced with selection
medium (DMEM with 10% FCS and 500 .mu.g/ml G418 (BRL). The G418
resistant colonies appeared after two to three weeks of selection.
The colonies were subcloned with limited dilution and examined by
radiolabelling and immunoprecipitation, ELISA, and
imnmunofluorescent staining for expression of recombinant proteins
as described [Marasco, W. A., et al., Proc. Natl. Acad. Sci. USA
90:7889 (1993)]. The Fd-protamine protein and kappa chain of F105
are expressed and secreted into the culture medium of
COS-Fab105-Protamine cells as detected by radiolabelling and
immunoprecipitation with anti-human IgG antibody See FIG. 3.
Purification of Fusion Proteins
The transformed COS cells (COS-Fab105-Protamine) were grown in
flasks with DMEM medium supplemented with 10% fetal calf serum
(FCS) and 500 .mu.g/ml of neomycin. After reaching confluence, the
cell cultures were replaced with fresh DMEM without FCS every three
days for two weeks. The collected culture medium was clarified by
centrifugation at 5000 rpm for 20 minutes at 4.degree. C., and then
concentrated using an Amico concentrator with membrane filter
molecular weight cut-off 10,000 dalton. The concentrated medium was
then loaded onto an affinity column coupled with anti-human IgG
kappa chain monoclonal antibodies (Kirkegaard & Perry
Inc.).
Preparation of the affinity column was made by mixing 2 mg of the
purified monoclonal antibody with 1 ml of wet beads of protein
A-sepharose CL-4B (Pharmacia Inc. Uppsala, Sweden) as described.
Briefly, protein-A-sepharose 4B beads were washed with PBS and then
mixed with purified antibodies in PBS at 4.degree. C. overnight.
The mixture was washed with 10 volumes of 0.2 M sodium borate (pH
9.0) and added with dimethypimelimidate to a final concentration of
20 mM. The mixture was stirred for 30 minutes at room temperature
on a rocker, washed once with 0.2 M ethanolamine (pH 8.0) and then
incubated for 2 hours at room temperature in 0.2 M ethanolamine on
a rocker. After final washing, the beads coupled with antibodies
were resuspended in PBS with 0.01% merthiolate.
The affinity column was washed with PBS, and loaded with the
concentrated culture medium. The medium passed through the column
followed with PBS washing until no protein was detected from the
elute. The column was washed with pre-elution buffer (10 mM
phosphate, pH 8.0) and eluted from the column with pH 2.4, 100 mN
glycine. The protein peak fractions were detected by Bradford
protein assay (Bio-Lab) and pooled together and dialyzed against
0.2 M NaCl. The DNA-binding portion of the fusion protein was
examined by incubation of the DNA-cellulose with the culture medium
of radiolabeled cells.
The transformed cell line (COS-Fab105-Protamine) was generated with
G418 (Gibco-BRL) selection after transfection with
pCMV-Fab105-Protamine DNA [Warrant, R. W., et al., Nature 271:130
135 (1978)]. The COS-Fab105 cell line was established as described
previously [Warrant, R. W., Nature 271, supra]. To examine
expressed proteins, the transformed cells were radiolabeled for 4
hours and precipitated with anti-human IgG (Southern Biotech) and
protein A-Sepharose 4B beads or with DNA-cellulose (Pharmacia) and
analyzed by SDS-PAGE as described previously [Chen,. S. -Y. et al.,
J. Virol. 65:5902 5909 (1991)]. To purify secreted Fab105-Protamine
in the serum-free medium, the culture medium of
COS-Fab105-Protamine cells is clarified, concentrated, and loaded
onto an affinity column of Protein-A-sepharose 4B beads coupled
with anti-human IgG monoclonal antibody, which was prepared
according to described methods [Winter, G., et al., Nature 349:293
299 (1992)]. The bound-proteins on the column were eluted by 100 mM
glycine (pH 7.5), and then concentrated and dialyzed against 0.20 M
NaCl solution. For ELISA, microtiter plates were coated with
recombinant gp120 (American Biotechnology Inc.) and incubated with
known concentration of Fab105 or Fab105-Protamine proteins followed
by incubation with anti-human IgG conjugated with alkaline
phosphatase (Sigma) [Warrant, R. W., et al., Nature 271,
supra].
FIG. 3 shows radiolabeling and immunoprecipitation of the expressed
fusion proteins. The transformed cell line (COS-Fab105-Protamine)
was generated as discussed above. The cells on 6-well plates were
continuously radiolabeled with .sup.35S-cysteine for 4 hours and
the culture medium of the cells was precipitated with either
anti-human IgG antibody (Southern Biotech) followed by
Sepharose-protein-A or with DNA-cellulose (Pharmacia). The samples
were analyzed by SDS-PAGE under reducing conditions. Lane 1,
COS-Fab105-Protamine precipitated with anti-human IgG; lane 2,
COs-vector precipitated with anti-human IgG and DNA-cellulose; lane
3, COS-Fab105-Protamine precipitated with DNA-cellulose; lane 4 and
5, COS-Fab105 precipitated with DNA-cellulose (4) or with
anti-human IgG (5).
The DNA-cellulose coprecipitated the Fd-protamine fusion proteins
and Kappa chain, but not the Fab105 fragment, suggesting that the
DNA-binding portion cf the Fd-protamine fusion protein maintains
its DNA binding ability and the fusion proteins are associated
together. The binding activity against HIV gp120, approximately 0.1
.mu.g/ml/24 hours, was detected in the culture medium of
COS-Fab105-Protamine cells by enzyme-linked immunosorbent assay
(ELISA), while no binding activity was observed in the medium of
vector-transformed cells.
The secreted recombinant fusion proteins were purified from
serum-free culture medium by using an affinity-column coupled with
anti-human IgG kappa chain monoclonal antibody (FIG. 4). The fusion
proteins bound to the column were eluted by 100 mM glycine (pH
2.4), concentrated and analyzed by SDS-PAGE under nonreducing or
reducing conditions following. As shown in FIG. 2, under the
reducing condition, two protein bands, corresponding to
Fd-protamine fusion protein and kappa chain appeared on the gel.
While under non-reducing conditions, the majority of the proteins
shifted to a higher molecular weight band, which likely represents
assembled Fab fragments. The specific binding activity of the
purified Fab105-Protamine to gp120, although slightly lower than
that of Fab105, was detected by ELISA.
FIG. 4 shows the purification and SDS-PAGE analysis of the
recombinant fusion protein.
The Fab105-Protamine fusion proteins in the culture medium were
purified by an affinity-column coupled with anti-human IgG kappa
chain monoclonal antibodies (Kirkegaard and Perry Lab) as
described. The proteins bound to the column were eluted by 100 mM
glycine (pH 2.4) and then concentrated and dialzyed against 0.20 M
NaCl solution. The purified proteins were analyzed by SDS-PAGE
under reducing or nonreducing conditions following Coomassie blue
staining.
Lane a, 100 ng (left), Lane a' 10 ng (right) of purified
Fab105-Protamine under the reducing conditions; Lane b 100 ng of
purified Fab105-Protamine under non-reducing conditions.
Binding activity to Gp120 of the purified fusion protein is shown
in FIG. 5.
ELISA plates coated with recombinant HIV-1 gp120 (American
Biotechnology, Inc.) were incubated with Fab105 or Fab105-Protamine
proteins followed by anti-human IgG conjugated with alkaline
phosphatase (Sigma). The binding activity to gp120 was detected at
OD.sub.405 after incubation with substrate (Bio-Lab). The data
shown are the mean values from duplicate determination. Lane a, 10
ng/ml of Fab105 or Fab105-protamine; Lane b, 1 ng/ml; Lane c, 0.1
ng/ml and Lane 3, 0.01 ng/ml. The first column in each lane is
Fab-105, while the second column is Fab-105-Protamine.
These results indicate that the Fab105-Protamine fusion proteins,
which are assembled and secreted into the culture medium, have
specific binding activity to HIV-1 gp120.
The DNA binding activity of Fab105-Protamine was examined by a
gel-shift assay [Wagner, E., et al., PNAS USA:89:6099 6103
(1992)].
DNA Binding Assay
Gel-shift assay was used to analyze the DNA binding activity of the
recombinant fusion proteins. The increased amounts of purified
fusion proteins in 0.2 N NaCl solution were mixed with a given
amount of DNA either radiolabeled or unlabeled in 0.2 N NaCl
solution. DNA radiolabelling with .sup.32P-dATP (Amrasham) was
performed using a nick translation kit (Promega). The protein-DNA
mixtures was allowed to stand at room temperature for 30 minutes
and filtered through 0.45 uM pore-size membrane to eliminate
DNA-protein precipitates, and then loaded onto 1.0% agarose gel for
electrophoresis at 1.times.TAE buffer. To analyze cytotoxicity of
the DNA-toxin expressor, the fusion protein-DNA mixtures were
dialyzed against the normal saline solution at 4.degree. C.
overnight before adding to cell cultures.
The DNA binding activity of Fab105-Protamine protein is shown in
FIG. 6.
DNA-binding ability of Fab105-Protamine was examined by gel
mobility-shift assay [Wu, G. Y., et al., J. Biol. Chem. 262:4429
4432 (1987)]. The HindIII/XbaI-cut DNA fragments of
pCMV-Fab105-Protamine was radiolabeled with .sup.32P-dATP using a
nick-translation kit (Pharmacia). 20 ng of labeled DNA for each
sample was incubated with increased amount of Fab105-Protamine
proteins in 0.20 N NaCl solution. DNA were incubated with Fab105
proteins as control. The whole plasmid DNA pCMV-Fab105-Protamine
(0.2 .mu.g each sample) were also incubated with increased amount
of pCMV-Fab105-Protamine proteins in 0.20 N NaCl (See, FIG. 7). The
samples were analyzed by electrophoresis on 0.8% agarose gels. For
autoradiography, the gel was dried, and exposed on X-ray film. FIG.
6: lane 1, DNA (5 ng) only; lanes 2 to 4, DNA (5 ng) with 0.5 ng
Fab-Protamine (2); with 1.0 ng Fab-Protamine (3); with 10 ng
Fab105-Protamine (4); lane 5, DNA (5 ng)/10 ng Fab105 as control.
FIG. 7, lane 1, DNA (0.2 .mu.g) only; lane 2, DNA (0.2 .mu.g)/2.0
.mu.g Fab105 control; lanes 3 to 6, DNA (0.2 .mu.g) with 0.1 .mu.g
Fab-Protamine (3); with 0.2 .mu.g Fab-Protamine (4); with 0.4 .mu.g
Fab-Protamine (5); with 0.6 .mu.g Fab-Protamine (6), lane 7,
Fab105-Protamine only (0.6 .mu.g); and lane 8, DNA (0.2 .mu.g) with
0.6 .mu.g of Fab105-Protamine/phenol extract before loading onto
the gel.
As shown in FIGS. 6 and 7, when increasing amounts of the fusion
proteins were mixed with the radiolabeled DNA fragments or whole
plasmid DNA, the decreasing amounts of DNA fragments or whole
plasmid DNA migrated into agarose gels and the DNA entered the
agarose gels migrated slower, while the DNA incubated with Fab105
proteins showed no significant change of its mobility in the
agarose gels. The binding activity of the fusion proteins to gp120
on the cell surface after coupling with DNA was further examined by
fluorescent activated cell sorting (FACS).
Binding ability of Fab105-Protamine-DNA complexes to GP120 on the
cell surface is shown in FIG. 8.
The HIV-infected or mock-infected Jurkat cells were incubated with
Fab105 or Fab105-Protamine protein-DNA complexes followed by
anti-human IgG Fab [Pastan, I., et al., Science 254:1173 (1992)]
conjugated with Fitc. The fluorescent staining on the cell surface
was then analyzed by FACS.
The DNA mobility-shift assay was performed as described [Wu, G. Y.,
et al., J. Biol. Chem. 262:4429 (1987); Wagner, E., et al., Proc.
Natl. Acad. Sci. USA 89:6099 (1992)]. Increased amounts of purified
fusion proteins were mixed with given amounts of DNA in the 0.2 M
NaCl solution. The mixtures standed at room temperature for 30
minutes and then filtered through 0.45 .mu.M membrane (Millipore)
before loaded onto 0.8% agarose gels for electrophoresis. To detect
the binding ability of fusion protein-DNA complexes to gp120 on the
cell surface, 1 .mu.g of purified Fab105-Protamine was mixed with
0.5 .mu.g of pCMV-Fab105 plasmid DNA in 100 .mu.l of 0.2 N NaCl for
30 minutes, and the mixtures were diluted to 1:20 at 0.9% N NaCl
solution and incubated with HIV-1 infected or uninfected Jurkat
cells followed by anti-human IgG-Fitc conjugates. Fab105 fragments
were used as a control. The fluorescent staining was then analyzed
by FACS.
As shown in FIG. 8, the HIV-1-infected cells reacted with either
Fab105 or the Fab105-Protamine-DNA complexes showed positive
staining, while uninfected cells incubated with the complexes
showed negative staining. The infected cells directly incubated
with conjugated antibody also showed negative staining (not shown).
Thus, the Fab105-Protamine fusion proteins maintain the binding
activity to gp120 after coupling with DNA molecules.
The encoding gene of PEA was selected to construct mammalian toxin
expression vectors due to the accumulated knowledge of the encoding
gene sequence-function relation [Gary, G. L, et al., Proc. Natl.
Acad. Sci USA, 81 supra; Allured, V. S., et al., Proc. Natl. Acad.
Sci. USA 83, supra; Siegall, C. B., et al, J. Biol. Chem. 264,
supra], PEA has several functional domains: Domain I is responsible
for cell recognition: domain II, for translocation of the toxin
cross membrane; and domain III, for adenosine diphosphate
(ADP)-ribosylation of elongation factor 2, the step actually
responsible for cell death [Allured, V. S., Proc. Natl. Acad. Sci.
USA 83, supra; Siegall, C. B., et al., J. Biol. Chem. 264, supra].
Two PEA mammalian expression vectors were designed and constructed
in which the domain III (mature PEA amino acid residues of 405 to
613) only (pCMV-PEAIII) or domain III and partial domain Ib
sequence (amino acids of 385 to 613) only (pCMV-PEAIb-III) was
placed under the control of CMV and T7 promoter.
Construction of Toxin Expression Vectors
A plasmid pJH8 containing the PEA encoding gene was obtained from
the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Md. 20852, having ATCC Depost No. 67208. See, FIG. 9 for
a schematic showing the PEA encoding domains. The DNA sequences
encoding the PEA catalytic fragment were obtained by PCR
amplification using pJH8 DNA as a template. To construct the toxin
expressor designated as pCMV-PEIII, an upstream primer (P1, (SEQ ID
NO:7) 5'TTTAAGCTTATGGGCGACGTCAGCTTCAGCACC-3') containing an
additional HindIII site and an initial codon followed by sequences
complimentary to the amino acids 405 to 411 of mature PEA and a
downstream primer (P-2, (SEQ ID NO:8)
5'-TTTTCTAGATTACTTCAGGTCCTCCGG-3') containing the sequence
complimentary to amino acids 609 to stop codon of PEA followed by
an additional XbaI site were used to amplify domain III of PEA. To
construct the toxin expresser pCMV-PEIbIII, an upstream primer (SEQ
ID NO:9) (P3 5'-TTTAAGCTTATGGCCGACGTGGTGAGCCTG-3'), corresponding
to amino acids 365 to 372 of PEA, and the downstream primer P-2
were used to amplify the partial domain Ib and domain III. The
amplified DNA fragments were purified with Geneclean kits (Bio 101
Inc.), digested with HindIII/XbaI and cloned into the pRc/CMV
expression vector (Invitrogen) under control of CMV promoter. The
resulting constructs were confirmed by DNA sequencing.
These constructs ensure that any expressed toxin fragments without
the recognition domain are nontoxic to surrounding cells unless
they are expressed inside a cell. To detect the toxin fragments
expressed from the vectors, the plasmids pCMV-PEAIII or
pCMV-PEAIbIII (See FIG. 9) were first transformed into BL21 (DE3)
expression bacterial hosts (Novagen), which inducibly express T7
DNA polymerase for transcription of the gene under the control of
T7 promoter. ADP-ribosylation activity was detected from the
transformed bacteria after induction (not shown). When the toxin
expressors were transfected into mammalian cells (COS-1 and HeLa)
using lipofectin [Chen, S. -Y., et al., J. Virol. 65:5902 5909
(1991)], toxin fragments were produced and cytotoxicity to the
transfected cells was observed (not shown). The pCMV-PEIbIII vector
which showed a higher level activity of ADP-ribosylation than
pCMV-PEIII was used for further experiments.
To investigate whether Fab105-Protamine can function as a gene
carrier to transfer the toxin expressor into target cells, the
purified Fab105-protamine fusion proteins were incubated with
pCMV-PEAIbIII plasmid DNA at 2:1 ratio (determined by titration) in
0.2 N NaCl solution to form fusion protein-DNA complexes (See FIGS.
10 12). HIV-1-infected Jurkat lymphocytes which were shown over 95%
positive by immunofluorescent staining with the antibody against
gp120 were used as target cells. The target cells were incubated
with the Fab105-Protamine-toxin expressor complexes, toxin
expressors only or Fab105-protamine proteins alone. Normal
lymphocytes were also incubated with these molecules as control.
After 48 hours of incubation, cell viability (FIG. 10), protein
synthesis (FIG. 11), and ADP-ribosylation activity (FIG. 12) in the
culture cells were examined.
The selective cytotoxicity of Fab105-Protamine-toxin Expressor
Complexes to HIV-Infected Cells are shown in FIGS. 10 12.
Jurkat cells were infected with HIV-1 virus and at day 10
postinfection the surface gp120 expression of the cells were
examined by immunofluorescent staining. The cells with over 95%
gp120 positive (0.5.times.10.sup.6) were incubated with the
DNA-fusion protein complexes, or fusion protein only, or DNA only
at 37.degree. C. for 48 hours.
FIG. 10 shows cell viability in the culture examined by Trypan Blue
Staining.
Percentage of viable cells were calculated from duplicate
determination.
FIG. 11 shows a protein inhibition assay, the cells
(0.5.times.10.sup.6) were replaced with leucine-free medium (See,
Allured, V. S., et al., Proc. Natl, Acad. Sci. USA 83:1320 (1986);
Pastan, I., et al., Cell 47:641 (1986); Siegall, C. B., et al., J.
Biol. Chem. 264:14256 (1989) and added with 4 .mu.ci
.sup.3H-leucine for 4 hours. The cells were centrifuged at 3,000
rpm for 5 minutes and lysed for scintillation counting.
FIG. 12 shows detection of ADP-ribosylation activity in the culture
cells [Collier, R. J., et al., J. Biol. Chem. 246:1496 (1971)]. The
cells (1.times.10.sup.6) were pelleted by centrifugation and lysed
in 4 M urea solution. The supernatants of the lysates were then
subjected to the ADP-ribosylation assay and PEA proteins
(Gibco-BRL) were used for positive control. The mean scintillation
counts of the samples are shown as calculated from duplicate
determination.
In FIGS. 10 12 lane a: normal Jurkat cells incubated with
Fab-Protamine-toxin expressor complexes (10 .mu.g
Fab105-Protamine/5 .mu.g expressor DNA, 10:5 .mu.g); lanes b to e:
HIV-infected Jurkat cells incubated with Fab-Protamine only (b);
with toxin expressor DNA only (C); with Fab105-Protamine-Toxin
expressor complexes (10:5 .mu.g) (d); with Fab105-Protamine-toxin
expressor complexes (5:2.5 .mu.g) (e); lane f:ADP-ribisylation
activity of 1.0 ng of urea-denatured PEA control.
As shown in FIGS. 10 12, after incubation with
Fab105-Protamine-toxin expressor complexes for 48 hours, the
HIV-1-infected cells showed a significant decrease of cell
viability (<1.5%) and protein synthesis ability (<0.2%);
while the cells incubated with toxin expressor or Fab105-protamine
alone only showed slightly decreased cell viability and protein
synthesis ability. In addition, uninfected lymphocytes showed no
significant decreases of cell viability and protein synthesis
ability after incubation with the Fab105-Protamine-toxin expressor
complexes. The observed selective cytotoxicity to HIV-infected
cells should be a result of ADP-ribosylation activity from
expressed toxin fragments since ADP-ribosylation activity, roughly
equal to 1.0 ng of PEA proteins, was detected from lysates of the
HIV-infected cells (1.times.10.sup.6) incubated with the complexes.
Thus, the Fab105-Protamine-toxin expressor complexes selectively
intoxicate HIV-1 infected cells in tissue culture.
A major obstacle of immunotoxins as efficacious agents in the
treatment of human cancer and other diseases is the host antibody
response to xenogeneic antibodies and toxin molecules.[Byers, V.
S., et al., Immunol. 65:329 (1988); Durrant, L. G., et al., Clin.
Exp. Immunol. 75:258 (1989); Pai, L. H., et al., J. Clin. Oncol.
9:2095 2103 (1991)]. The utilization of humanized murine or human
antibodies may solve the problem of targeting moiety [Rybak, S. M.,
et al., Proc. Natl. Acad. Sci. USA 89:3165 3169 (1992)], but for
highly immunogenic toxin moiety, the problem still remains. In this
study we demonstrate that the anti-gp120 Fab105-Protamine fusion
proteins can serve as a gene carrier to deliver toxin-expressor
plasmid DNAs into HIV-1-infected cells by receptor-mediated
endocytosis, resulting in selective killing of the target cells.
The extreme potency of the toxin molecules compensates for the low
efficiency of the receptor-mediated gene delivery [Wu, G. Y., et
al., J. Biol. Chem. 262:4429 4432 (1987); Wagner, E., et al., PNAS
USA 89:6099 6103 (1992)] to efficiently achieve the therapeutic
goal. Since that antibody molecules or ligands (targeting moiety)
and DNA-binding moiety of bifunctional fusion proteins can be of
human origin, and the toxin expressor DNAs are very weakly or non
immunogenic, the whole protein-toxin expressor complexes will be
weakly immunogenic. Therefore, these complexes should be able to be
repeatedly administered into patients without development of
significant antibody response. Furthermore, the bifunctional
recombinant fusion proteins as a gene carrier also have the
advantage over chemically linked ones [Wu, G. Y., et al., J. Biol.
Chem. 262:4429 4432 (1987); Wagner, E., et al., PNAS USA 89:6099
6103 (1992)], such as efficient production, and potentially better
binding activity. In summary, this gene therapy form of
immunotoxins, termed herein "stealth immunotoxins" has significant
advantages over currently described immunotoxins for treatment of
cancers, and other diseases. Moreover, the anti-gp120
Fab105-Protamine-toxin expresser complexes have selective toxicity
to HIV-1-infected cells which also represent a novel therapeutic
agent for AIDS treatment.
All references described herein are incorporated herein by
reference.
It is evident that those skilled in the art given the benefit of
the foregoing disclosure may make numerous other uses and
modifications thereof and departures from the specific embodiments
described herein without departing from the inventive concepts, and
the present invention is to be limited solely by the scope and
spirit of the appended claims.
SEQUENCE LISTINGS
1
9 8 amino acidsamino acidunknownunknown1Lys Lys Lys Tyr Tyr Lys Leu
Lys1 536 base pairsnucleic acidunknownunknown2TTTGAATTCA AGCTTACCAT
GGAACATCTG TGGTTC 3633 base pairsnucleic
acidunknownunknown3GGTACCGAAT TCTCTAGAAC AAGATTTGGG CTC 3336 base
pairsnucleic acidunknownunknown4GGTACCGAAT TCTCTAGAAT GGCCAGGTAC
AGATGC 3648 base pairsnucleic acidunknownunknown5TTTAGGATCC
TTAACAACAC CTCATGGCTC TCCTCCGTGT CTGGCAGC 4878 base pairsnucleic
acidunknownunknown6TTAATTGCGG CCGCTTAGTG TCTTCTACAT CTCGGTCTGT
ACCTGGGGCT GACACCTCAT 60GGCTCTCCTC CGTGTCTG 7833 base pairsnucleic
acidunknownunknown7TTTAAGCTTA TGGGCGACGT CAGCTTCAGC ACC 3327 base
pairsnucleic acidunknownunknown8TTTTCTAGAT TACTTCAGGT CCTCCGG 2730
base pairsnucleic acidunknownunknown9TTTAAGCTTA TGGCCGACGT
GGTGAGCCTG 30
* * * * *